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Cailing Liu, Yuming Chen, Ula Jurkunas; Decline in DJ-1 Leads to Decreased Nuclear Translocation of Nrf2 and Results in p53-mediated Apoptosis of Human Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2196.
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Dowregulation of DJ-1 and decreased nuclear localization of nuclear factor erythroid-derived 2-like 2 (Nrf2) has been detected in Fuchs endothelial corneal dystrophy endothelium. DJ-1 has been reported to stabilize Nrf2, which binds to antioxidant response element in the nucleus to protect cells from apoptosis. In this study, we investigated the effect of DJ-1 downregulation on Nrf2 nuclear translocation, Nrf2-associated protein regulation, and corneal endothelial cell susceptibility to oxidative stress.
An immortalized normal human corneal endothelial cell line (HCECi) was transfected with 50 nM of siRNA specific to the human DJ-1 gene using Lipofectamine. Scrambled siRNA was used as control. Nrf2 and p53 levels in nuclear and cytosolic extracts were evaluated by western blotting. Nrf2-associated proteins such as Cul3 and Keap1 were detected by immunoprecipitation (IP) with anti-Nrf2 antibody, followed by western blotting with target antibodies. Oxidative stress was induced by exposing HCECi cells to a UVA broadband lamp with the fluence of 10 J/cm2. Cell pellets were harvested for western blotting with anti-caspase 3 and p53 antibodies, while supernatants were collected for a cell viability assay.
Despite similar levels of cytoplasmic Nrf2, nuclear Nrf2 protein levels decreased by 2.2-fold (p=0.04) in DJ-1 siRNA-treated HCECi cells as compared to scrambled siRNA-treated cells. IP studies detected Nrf2 association with Cul3 and Keap1 with an increase in Cul3-Nrf2 complex in DJ-1 siRNA-treated cells relative to controls. UVA irradiation led to an 8.5% increase in cell death in DJ-1 siRNA-treated cells as compared to controls. Moreover, downregulation of DJ-1 led to a 16.4% increase in active caspase 3 levels. This effect was augmented by UVA treatment, which led to a 28.6% increase in caspase 3 as compared to controls. Downregulation of DJ-1 resulted in an increase in cytoplasmic p53 levels, while UVA irradiation resulted in a 17.7% increase in total p53 levels in DJ-1 siRNA treated cells as compared to controls.
Downregulation of DJ-1 impairs nuclear translocation of Nrf2 potentially by targeting Nrf2 for degradation through Cul3 and Keap1 pathways. Decline in DJ-1 levels leads to heightened cell susceptibility to UVA-induced apoptosis and activates p53-dependent pathway in corneal endothelium.
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